MUTATIONAL ANALYSIS OF THE CATALYTIC SUBUNIT OF THE YEAST VACUOLAR PROTON-TRANSLOCATING ATPASE

In order to generate a set of tools for probing structure-function rel ationships in the catalytic subunit of the yeast vacuolar H+-ATPase, t he gene encoding this subunit (VMA1) was randomly mutagenized. Mutant plasmids unable to complement the growth defects of yeast cells lackin g an intact VMA1 gene were isolated and sequenced. Eight different mut ant alleles of VMA1 were examined for levels of the catalytic subunit and other subunits of the enzyme, assembly of the ATPase complex, targ eting to the vacuolar membrane, and concanamycin A-sensitive ATPase ac tivity. The mutations S811P and E740D resulted in mutant enzymes that assembled fully but were incapable of ATP hydrolysis, and the mutation E785G generated a similar but somewhat less severe phenotype (17% of the ATPase activity of wild-type vacuoles). When MgATP-dependent strip ping of the peripheral subunits by 100 mM KNO3 was examined in these t hree mutants, only the E785G mutant exhibited significant stripping, s uggesting that ATP hydrolysis, even at relatively low levels, generate s a conformation susceptible to dissociation. Plasmids containing the mutations E751G and F752S partially complemented the growth defects an d resulted in partial defects in ATPase activity that appear to reflec t reduced catalytic efficiency, Partial defects in growth and ATPase a ctivity were also seen in the Y797H mutant, but this mutation caused a n assembly defect manifested as a preferential loss of two of the peri pheral subunits of the enzyme. The phenotypes of these mutants are int erpreted in the context of homologies with other V-type and F-type ATP ases.